Endogenous norepinephrine regulates blood flow to the intact rat tibia.

The goal of our study was to determine if endogenous norepinephrine (NE) has a role in the regulation of basal blood flow to intact bone. The experimental plan was to measure bone blood flow before and after pharmacological blockade of alpha-adrenergic receptors. A significant increase in blood flow after receptor blockade would suggest that endogenous norepinephrine exerts a tonic constrictor effect on the vessels supplying blood to the bone. Mature, male rats were anesthetized with Inactin. Arterial blood pressure and left tibia blood flow (laser Doppler flowmetry) were measured. A cannula was inserted into the right iliac artery and advanced to the aortic bifurcation to deliver drugs into the left hindlimb circulation, including the left tibia vasculature. Bolus injection of norepinephrine caused a dose-dependent decrease in bone blood flow (30-40%). Blockade of alpha-adrenergic receptors with phentolamine or phenoxybenzamine attenuated by more than 50% the norepinephrine-induced decrease in bone blood flow. In separate rats that had not received exogenous norepinephrine, injection of phentolamine alone decreased bone vascular resistance by 34+/-3%. Similarly, phenoxybenzamine decreased resistance by 25+/-4%. These results are consistent with the conclusion that alpha-adrenergic receptors mediate a significant constriction of blood vessels which participate in the partial control of basal blood flow to the intact rat tibia.

Posterior lumbar interbody cages do not augment segmental biomechanical stability.

The effect on stiffness of installing posterior threaded interbody cages at LA-L5 was evaluated using fresh human cadaveric spine specimens. The cages did not increase spine stiffness significantly in any tested range of motion. Supplemental posterior pedicular screw/rod instrumentation, however, significantly increased stiffness. The assertion that use of cages as isolated posterior implants improves stability may be invalid.

Bone blood flow and vascular reactivity.

Blood flow is essential for normal bone growth and bone repair. Like other organs, the regulation of blood flow to bone is complex and involves numerous physiologic mechanisms including the sympathetic nervous system, circulating hormones, and local metabolic factors. Our studies addressed the following questions: (1) Which endogenous vasoconstrictor agents regulate in vivo blood flow to bone? (2) Does a decrease in bone vascular reactivity to vasoconstrictor hormones account for the increase in blood flow during bone healing? (3) Does the endothelium influence bone arteriolar function? An intact bone model was developed in the rat to assess hormonal regulation of in vivo bone blood flow and in vivo bone vascular reactivity. An isolated, perfused bone arteriole preparation was employed to characterize the responsiveness of small resistance-size arterioles (diameter < 100 microm) to vasoconstrictor hormones and to evaluate the role of the vascular endothelium to modulate vascular smooth muscle reactivity. Our results indicate that: (1) though exogenous endothelin is a potent constrictor of the in vivo bone vasculature, endogenous endothelin does not actively regulate in vivo blood flow; (2) the increase in blood flow to a bone injury site is not due to a decrease in bone vascular sensitivity to norepinephrine, and (3) isolated bone arterioles of young rats are very sensitive to vasoconstrictor hormones but exhibit only modest endothelium-mediated vasodilation.

Survival and induction of SOS in Escherichia coli treated with cisplatin, UV-irradiation, or mitomycin C are dependent on the function of the RecBC and RecFOR pathways of homologous recombination.

Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The antitumor effects of these drugs are due to the formation of bifunctional adducts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study the cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilitate the production of a molecular signal for induction of the SOS response. Strains used in the SOS chromotest have a fusion of lacZ with the sfiA (sulA) gene so that the amount of SOS inducing signal, which is modulated by the ability of the cell to repair DNA, is measured by assaying beta-galactosidase activity. SOS induction in a strain proficient in homologous recombination (HR) was compared with that in isogenic strains deficient in HR due to a blocked RecBC pathway caused by a recB mutation or a blocked RecFOR pathway caused by a recO mutation. The effect of cisplatin treatment in a uvrA mutant strain blocked at the first step of NER was compared with that in an isogenic strain proficient in NER. Cellular resistance was measured as percent colony forming units (cfu) for cells treated with increasing doses of cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demonstrated by decreased survival in mutants with the recB mutant being more sensitive than the recO mutant. SOS induction levels were elevated in the sensitive recB strain relative to the HR proficient strain possibly due to stalled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act at daughter strand gaps in newly synthesized DNA to mediate production of the signal for SOS induction. Proficiency in NER was necessary for both survival and high levels of SOS induction in cisplatin treated cells.

Outcome analysis of coronary artery bypass grafting: minimally invasive versus standard techniques.

Minimally invasive coronary artery bypass grafting (MIDCAB) procedures are purported to result in improvements in patient management over standard techniques. A comparative study was performed on risk-stratified patients treated with either technique. Following institutional review board approval, a retrospective random chart review was conducted on 27 MIDCAB and 37 standard coronary artery bypass grafting (CABG) patients who were operated on over a 12-month period at the University of Nebraska Medical Center. Risk stratification was accomplished by dividing the two patient populations, MIDCAB and 'standard', into one of four subgroups based on a preoperative risk score. Risk stratification was achieved by dividing the patient populations into one of four subgroups: good, fair, poor and high risk. Both groups received similar operations and surgical interventions, except for the inclusion of cardiopulmonary bypass (CPB). Approximately 200 parameters were collected and analyzed in the following categories: anthropometric, operative and postoperative outcomes. The MIDCAB group had a significantly lower number of vessels bypassed (2.0+/-0.7 vs 3.4+/-0.9, p < 0.0001). Total postoperative blood product transfusions trended higher in the standard group (6.1+/-12.6 U) when compared to the MIDCAB patients (2.3+/-5.5 U, p < 0.15), although not statistically significant. Postoperative inotrope use was significantly less in the MIDCAB group (19% vs 59%, p < 0.002). Ventilator time in the MIDCAB group was 10.5+/-5.4 h vs 15.0+/-12.3 h in the standard group (p < 0.07). The MIDCAB group had an overall greater length of stay, but was only statistically different within the poor-risk subgroup (12.2+/-10.7 vs 7.5+/-3.9, p < 0.04). The results of this study show that when CPB is not utilized in treating patients undergoing CABG procedures, the benefits in regards to patient outcomes are unclear. This necessitates the need for further work when comparing outcomes for risk-stratified patients.

Cardioplegia flow dynamics in an in vitro model.

The flow of fluids in extracorporeal circuits does not conform to conventional Poiseuille mechanics which confounds calculating cardioplegia (CP) flow distribution. The purpose of this study was to quantify CP flow dynamics in a model simulating coronary atherosclerosis across varying sized restrictions. An in vitro preparation was designed to assess hydraulic fluid movement across paired restrictions of 51, 81 and 98% lumen reductions. Volume data were obtained at variable flow, temperature, viscosity and pressure conditions. CP delivered through 14- and 18-gauge (GA) conduits at 8 degrees C and 100 mmHg infusion pressure revealed that both four to one and crystalloid CP solutions had significantly less total percentage flow through the 14-GA conduit, p < 0.0001 and p < 0.001, respectively. Overall, 4:1 CP exhibited the most favorable fluid dynamics at 8 degrees C in that it delivered the highest percentages of total CP flow through the smaller lumen conduit. At both 8 degrees C and 37 degrees C delivery, blood CP resulted in the least homogeneous fluid distribution at all delivery parameters. The results in relation to blood viscosity indicate that, although the 8 degrees C blood CP had a significantly greater viscosity than 37 degrees C blood CP, it did not produce an effect in fluid distribution. These data show that increasing the cardioplegic solution hematocrit causes an inhomogeneous fluid distribution regardless of delivery temperature or infusion pressure.

Removal of hepatocarcinoma cells from blood via cell washing and filtration techniques.

Utilization of autotransfusion during tumor resection remains controversial due to viability of carcinoma cells remaining in collected blood. The purpose of this study was to evaluate autotransfusion techniques combined with leukocyte depleting filters (LDF) for removal of hepatocarcinoma cells from autotransfusate. An in vitro model was created by contaminating expired human erythrocytes with cultured hepatocarcinoma (HEP G2) cells. Autotransfusion devices evaluated were Cobe BRAT2, Sorin STAT-P, and Fresenius CATS. Autotransfusate collected from varying processing conditions were filtered using the Pall Leukoguard RS or Pall Purecell RCQ LDF. Carcinoma concentrations were quantified via Coulter Counter technology. The CATS exhibited higher concentrations of cancer cells in the autotransfusate prior to washing, a 449% increase. This was significantly higher than either the BRAT2 or STAT-P, 350% and 315% respectively. Post washing HEP G2 concentrations in the BRAT2 were significantly higher than the STAT-P and CATS. Doubled wash volumes removed more HEP G2 cells in all trials, reaching statistical significance only in the CATS. LDF resulted in a significant 75% reduction of HEP G2 cells, with no difference between filters. While combination use of autotransfusion devices and leukocyte depleting filters did result in a product with concentrated hematocrit, no technique removed all hepatocarcinoma cells.

Effect of fluvoxamine therapy on the activities of CYP1A2, CYP2D6, and CYP3A as determined by phenotyping.

OBJECTIVE: To determine the effect of 150 mg/day fluvoxamine on the activities of CYP1A2, CYP2D6, CYP3A, N-acetyltransferase-2 (NAT2), and xanthine oxidase (XO) by phenotyping with caffeine, dextromethorphan, and midazolam. METHODS: Oral caffeine (2 mg/kg), oral dextromethorphan (30 mg), and intravenous midazolam (0.025 mg/kg) were administered to 10 white male volunteers every 14 days for 4 months and to 10 white premenopausal female volunteers during the midfollicular and midluteal phases of the menstrual cycle for 4 complete cycles (8 total phenotyping measures). The first 6 phenotyping measures were used to establish baseline activity. Subjects were given 150 mg/day fluvoxamine for the fourth month or cycle of the study. Enzyme activity for CYP1A2, CYP2D6, NAT2, and XO was expressed as urinary metabolite ratios. Midazolam plasma clearance was used to express CYP3A activity. RESULTS: No difference between baseline and weeks 2 and 4 of fluvoxamine therapy was observed for NAT2 or XO metabolite ratios. For CYP1A2, CYP2D6, and CYP3A phenotypes, significant differences existed between baseline and fluvoxamine therapy. For CYP1A2, the mean urinary metabolite ratio (+/-SD) was 7.53 +/- 7.44 at baseline and 4.30 +/- 2.82 with fluvoxamine ( P = .012). Mean CYP2D6 molar urinary dextromethorphan ratios before and after fluvoxamine therapy were 0.00780 +/- 0.00694 and 0.0153 +/- 0.0127, respectively (P = .011). Midazolam clearance decreased from 0.0081 +/ 0.0024 L/min/kg at baseline to 0.0054 +/- 0.0021 L/min/kg with therapy (P = .0091). For CYP1A2, CYP2D6, and CYP3A, fluvoxamine therapy changed the phenotyping measures by a median of -44.4%, 123.5%, and -34.4%, respectively. CONCLUSIONS: We concluded that fluvoxamine may cause significant inhibition of CYP1A2, CYP2D6, and CYP3A activity. This metabolic inhibition may have serious implications for a variety medications.

Quantification of 3-month intraindividual variability and the influence of sex and menstrual cycle phase on CYP3A activity as measured by phenotyping with intravenous midazolam.

OBJECTIVE: Intraindividual variability and the effects of sex and menstrual cycle phase on CYP3A activity were evaluated by phenotyping with use of midazolam as the probe drug. METHODS: Midazolam (0.025 mg/kg) was administered intravenously to 10 white male volunteers every 14 days for 3 months and to 10 white premenopausal female volunteers during the midfollicular and midluteal phases of the menstrual cycle for 3 complete cycles. Serum was collected for a 6-hour period, and enzyme activity was represented by midazolam plasma clearance. RESULTS: No difference in clearance was observed during the menstrual cycle phases. Mean +/- SD midazolam clearance was 0.00816 +/- 0.00252 L/min/kg during the midfollicular phase and 0.00818 +/- 0.00224 during the midluteal phase (P = .96). When the menstrual cycle phases were combined, mean midazolam clearance in women was 0.00817 +/- 0.00235 L/min/kg. Mean male midazolam clearance was 0.00766 +/ 0.00167 L/min/kg. There was no difference in midazolam clearance between men and women (P = .68). Coefficients of variation (CV%) for the 6 phenotyping visits were calculated and the median midazolam clearance CV% (25th to 75th percentile) was 9.75% (8.40% to 11.5%). CONCLUSIONS: Because no significant differences in midazolam clearance were noted between menstrual cycle phases or between sexes, pharmacokinetic and clinical investigations of CYP3A activity in adults may not require stratification on the basis of menstrual cycle phase or sex.

A comparison of the genotoxic effects of carboplatin and cisplatin in Escherichia coli.

cis-Diammine(1,1,-cyclobutanedicarboxylato)platinum(II) (carboplatin) is a second generation platinum anticancer agent with antineoplastic properties like that of its parent compound, cis-diamminedichloroplatinum(II) (cisplatin) but with substantially less deleterious side effects in treated patients with cisplatin. We compared their genotoxic effects in Escherichia coli and found carboplatin to be less cytotoxic (measured as loss of colony forming ability) that cisplatin in that equitoxic doses required greater than 60 time more carboplatin. However, solutions of carboplatin containing chloride ion became more cytotoxic to E. coli after a 24 h incubation period than similar freshly made solutions. Two platinum conversion products which were neither present in freshly made solutions nor in solutions lacking chloride were resolved by thin-layer chromatography (TLC). One of the conversion products migrated like cisplatin and its occurrence in carboplatin solutions was associated with cisplatin-like properties, enhanced cytotoxicity and ability to induce the SOS responses in E. coli. The SOS-inducing abilities were determined by induction of a sulA::lacZ fusion. Likewise, adducts formed in end-labeled oligonucleotides treated with carboplatin appeared identical to those caused by cisplatin when carboplatin was preincubated in chloride-containing solutions but not by carboplatin in freshly made solutions. It is likely that responses evoked by carboplatin in biological systems are partly due to activation of carboplatin by its conversion of cisplatin.

Evaluation of drug interactions in intact hepatocytes: Inhibitors of terfenadine metabolism.

Terfenadine has been associated with several adverse drug interactions and it was of interest to develop in vitro systems to explain and predict such interactions. The metabolism of terfenadine was studied using intact hepatocytes from primary human and rat hepatocyte cultures, and the immortalized human hepatoma cell line HepG2. Rates and routes of biotransformation were analysed by HPLC. Terfenadine was extensively metabolized by all three cell culture systems during exposure periods ranging from 4 to 24 hr. Human and rat hepatocytes and HepG2 cells formed products of C-oxidation (an acid metabolite and its precursor alcohol metabolite). Human hepatocytes also formed the N-dealkylation product azacyclonol. Several cytochrome P4503A (CYP3A) substrates and inhibitors were evaluated for their ability to inhibit terfenadine biotransformation. In rat hepatocytes, ketoconazole, erythromycin and troleandomycin failed to inhibit; in HepG2 cells, only ketoconazole potently inhibited terfenadine metabolism. In human hepatocytes, ketoconazole, itraconazole, erythromycin, troleandomycin, cyclosporin and naringenin inhibited terfenadine metabolism. The results suggest that human hepatocytes may be a useful system for screening for inhibitors of terfenadine metabolism.

Polybrominated biphenyl induction of cytochrome P450 mixed function oxidase activity in primary rat and human hepatocytes.

Polybrominated biphenyl (PBB) is an industrial chemical and environmental contaminant with known incidence of significant human exposure. PBB has been studied in laboratory animals and found to have significant toxicological effects as well as being a potent inducer of hepatic cytochrome P450 mixed function oxidase (MFO) activity. As part of our program to compare the response of laboratory animals and humans to industrial and environmental toxicants, we studied the effect of a major component of commercial PBB mixtures, 2,2',4,4',5,5'-hexabromobiphenyl (HBB), on MFO induction in primary cultures of human and rat hepatocytes. MFO induction was evaluated by measuring the deethylation of 7-ethoxycoumarin by intact hepatocytes. Rat hepatocytes were found to be highly susceptible to HBB induction of 7-ethoxycoumarin O-deethylase (ECOD) activity, with significant induction observed at the lowest concentration tested of 10(-8) M. Human hepatocytes were found to have a higher threshold for HBB induction of ECOD activity than rat hepatocytes. The lowest concentration of HBB required for ECOD induction observed for human hepatocytes was 10- to 1000-fold higher (10(-7), 10(-6), 10(-5) M for the four human samples) than that found in rat hepatocytes. Future mechanistic investigation of this observed difference in sensitivity towards PBB between rat and human hepatocytes may aid the extrapolation of human health risk from toxicological data obtained from laboratory animals.

Arginine transport in human liver. Characterization and effects of nitric oxide synthase inhibitors.

OBJECTIVE: Arginine transport was characterized and studied in human liver. SUMMARY BACKGROUND DATA: Plasma arginine uptake may regulate hepatocyte intracellular availability and the subsequent biosynthesis of nitric oxide (NO), but little is known about arginine transport across the human hepatocyte plasma membrane. METHODS: The authors characterized plasma membrane transport of 3[H]-L-arginine in hepatic plasma membrane vesicles (HPMVs) and in hepatocytes isolated and cultured from human liver biopsy specimens. They also studied the effects of the NO synthase inhibitors omega-nitro-L-arginine methyl ester (L-NAME) and N-methyl-arginine (NMA) on arginine transport in HPMVs and in cultured cells. RESULTS: Arginine transport was saturable, Na(+)-independent, temperature and pH sensitive, and was inhibited by the naturally occurring amino acids lysine, homoarginine, and ornithine (System y+ substrates). Arginine transport by both vesicles and cultured hepatocytes was significantly attenuated by NO synthase inhibitors, suggesting that the arginine transporter and the NO synthase enzyme may share a structurally similar arginine binding site. Dixon plot analysis showed the blockade to occur by competitive, rather than noncompetitive, inhibition. In vivo treatment of rats with lipopolysaccharide (LPS) resulted in a twofold stimulation of saturable arginine transport in the liver. This LPS-induced hepatic arginine transport activity was also inhibited by L-NAME. These data indicate that arginine transport by human hepatocytes is mediated primarily by the Na(+)-independent transport System y+. CONCLUSIONS: Besides inhibition of the NO synthase enzyme, the ability of arginine derivatives to block NO production may also be due to their ability to competitively inhibit arginine transport across the hepatocyte plasma membrane. The use of selective arginine derivatives that compete with arginine at the plasma membrane level may be a metabolic strategy that can be used to modulate the septic response.

A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids.

A simple and highly reproducible method was established for the culturing of adult rat and human hepatocytes as multicellular aggregates (spheroids). Purified rat and human liver parenchymal cells were cultured on nontissue culture (bacteriological) polystyrene petri dishes on a rotating platform. After an overnight incubation, the cells were found to form multicellular aggregates. The aggregates became spheroidal in shape after several days in culture. Histological sections of the spheroids showed an organized structure consisting of squamated cells on the outermost layer and cuboidal cells in the interior. Cellular structures characteristic of hepatocytes in the liver in vivo including bile canaliculi, peroxisomes, Golgi bodies, abundant mitochondria, and rough and smooth endoplasmic reticulum were observed with electron microscopy. The spheroids were found to be viable up to the longest time studied of approx. 1 month in culture as demonstrated by their adherence and growth on collagen-coated substratum. The morphological resemblance between hepatocytes cultured as spheroids and the liver in vivo suggests that the spheroids may be a useful in vitro experimental model of the liver. Our simple method should allow hepatocytes to be cultured as spheroids easily in any laboratory equipped for cell culture. Our study here also is the first to report the culturing of human hepatocytes as spheroids.

Comparison of rat liver parenchymal and nonparenchymal cells in the activation of promutagens.

While the liver consists of both parenchymal cells (PC) and nonparenchymal cells (NPC), virtually all studies on promutagen activation have been performed using PC. To evaluate the comparative roles of PC and NPC in promutagen activation, we cocultivated a cell line generally considered to have an insignificant level of xenobiotic metabolism, Chinese hamster ovary (CHO) cells, with either PC, NPC, or a combination of both. The mixed culture was treated with two promutagens: dimethylnitrosamine (DMN) and 3-methylcholanthrene (3-MC). The induction of 6-thioguanine resistant mutants was evaluated using the well-established CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Activation of promutagens, as indicated by an increase in mutant frequency in CHO cells, was observed only when the PC were present with the CHO cells during the treatment period. No activation was observed with NPC. Coculturing of PC and NPC yielded essentially the same results as PC alone. P-450 mixed function monooxygenase activity measured by the 7-ethoxycoumarin-O-deethylase assay further substantiates that PC had a significantly higher xenobiotic metabolism activity than NPC. Our study therefore indicates that PC, not NPC, are the major cell population in the liver responsible for the activation of promutagens.

Recombinant fusion proteins of protein A and protein G with glutathione S-transferase as reporter molecules.

The regions encoding the IgG-binding domains of protein A (PA) and protein G (PG) were cloned into the bacterial expression vector pGEX. Both proteins were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (PA-GST and PG-GST) and were found to be soluble, abundant and easily purified in one step from the bacterial lysate by affinity chromatography on immobilized glutathione. Yields of 50 mg/litre of cultures were obtained. Both purified fusion proteins were shown to be functional in a variety of immunochemical procedures. In radial diffusion tests, PA-GST precipitated IgG from human, squirrel monkey, rabbit, dog, cat and pig but not mouse, sheep, goat, cow, horse or chicken. PG-GST formed precipitin bands with IgG from human, rabbit, mouse, pig, sheep, goat, cow and horse but not squirrel monkey, dog, cat and chicken IgG. The fusion proteins were shown to function as effective detection reagents in ELISA and Western blotting. Glutathione agarose beads with bound fusion protein were shown to be useful for immunoprecipitation.

Mass spectral investigations on trichothecene mycotoxins. VIII--Thermospray ionization and collisionally activated dissociation of macrocyclic trichothecenes.

Several structurally similar, biologically active macrocyclic trichothecenes were ionized under thermospray conditions followed by collisionally activated dissociation of the ammonium adducts. Most of the observed daughter ions were formed by bond cleavage at the exocyclic ester bridges. Compounds with similar ester bridges formed several common daughter ions and underwent similar neutral losses. Fragmentation pathways proposed for the dissociation of the adducts were confirmed from the corresponding neutral loss spectra. Simple experiments designed for the sequential monitoring of the characteristic daughter ions were used for the rapid, direct and accurate analysis of macrocyclic trichothecenes in real samples. The primary drawback of the method is its inability to distinguish between isomers.